白鳍鲨SBP1基因的克隆、表达及抗癌活性分析

Molecular Cloning,Expression,and Anti-Cancer Activity of SBP1 Gene in Whitetip Reef Shark

硒结合蛋白1(selenium-binding protein 1,SBP1)在多种癌症中起关键作用。目前对SBP1的研究大多集中在模式生物上,对未知基因组物种的SBP1知之甚少。该研究前期基于白鳍鲨体内高硒含量特性及SBP1肽段信号,首次克隆和测序分析了白鳍鲨SBP1的cDNA序列。白鳍鲨SBP1基因的cDNA全长为3 064 bp,开放阅读框为1 419 bp,编码472个氨基酸。白鳍鲨SBP1蛋白序列中组氨酸残基共15个,占比3.2%;其含有2个CxxC、2个HxD和3个HxxH保守区域。通过SWISS-MODEL构建白鳍鲨SBP1三维结构模型,推测定位于β-折叠与连接各结构环交界处的第56位丝氨酸(Ser56)为硒结合位点。系统进化树分析表明,白鳍鲨和象鲨的SBP1同源性最高。在人正常肝细胞HL-7702、人肝癌细胞HepG2和人宫颈癌细胞HeLa中分别转染重组子MYC-SBP1和空载体pcDNA3.1-MYC后,使用Cell Counting Kit-8和CFDA SE细胞增殖示踪荧光探针研究鲨鱼SBP1的抗癌活性。研究发现,无论培养基中是否添加Na_(2)SeO_(3),在HeLa和HepG2癌细胞中表达重组蛋白SBP1都会显著抑制细胞增殖。相反,表达SBP1组将促进正常HL-7702细胞增殖。该研究推测硒的化学预防作用与SBP1的功能高度相关。在某种程度上,可能是SBP1蛋白(而不是硒)在预防癌症中起着更关键的作用。

SBP1 (selenium-binding protein 1) plays a key role in a variety of cancers.At present,most researches have focused on model organisms,while few studies have explored the SBP1 of unknown genome species.In the early stage of this study,based on the discovery of the high selenium content and the SBP1 peptide signal in the whitetip shark,the SBP1 cDNA sequence of the whitetip reef shark was first cloned and sequenced.The full-length cDNA of the whitetip reef shark SBP1 gene is 3 064 bp and contains a 1 419 bp open reading frame that encodes 472 amino acids.There are 15 histidine residues within its protein sequence,accounting for 3.2%.It contains two CxxC,two HxD and three HxxH conserved regions.The three-dimensional structural model of SBP1 was constructed by SWISS-MODEL,and it was deduced that the 56th serine (Ser56) located at the junction between the β-sheet and structural loop was the selenium binding site.Phylogenetic tree analysis shows that whitetip shark and elephant shark have the highest SBP1 homology.After transfecting the recombinant MYC-SBP1 and the empty vector pcDNA3.1-MYC into HL-7702,HepG2 and HeLa cells respectively,Cell Counting Kit-8 and CFDA SE cell proliferation tracer fluorescent probe were used to investigate the anti-cancer function of shark SBP1.It was found that whether or not Na_(2)SeO_(3) was added in medium,the expression of the recombinant protein SBP1 in HeLa and HepG2 cancer cells would significantly inhibit cell proliferation.In contrast,the SBP1 expression group promotes the proliferation of normal HL-7702 cells.This study speculates that the chemopreventive effect of selenium is highly related to the function of SBP1.To some extent,it may be SBP1 protein rather than selenium plays a more critical role in cancer prevention.